Costs of musculoskeletal conditions represent an average of 3% of the gross domestic product of developed countries, consuming an estimated $254 billion annually in the V.S.. Engineering of natural musculoskeletal tissues represents a promising new approach to expand the range of conditions that can be effectively treated. This proposal focuses on development of a new approach for regenerating natural skeletal tissues, with an emphasis on temporally controlling the activity of adult stem cells using protein growth factors. The guiding hypothesis of this work is that engineered growth factors can be included into growing layers of an inorganic matrix, resulting in sequential growth factor delivery upon material dissolution. Growth factors will be tagged with a mineral binding sequence for inclusion into calcium-based minerals, and subsequent matrix dissolution will enable sequential delivery. Delivery of each growth factor will be designed to elicit a distinct mesenchymal stem cell (MSC) response, allowing for temporal control over early generation of new bone tissue. In a first demonstration of the utility of our approach, we will release a mitogenic factor (FGF-2) and an osteogenic factor (BMP-2) in sequence. Specific Aim 1will develop and characterize a method for growing calcium-based minerals within a macroporous alginate hydrogel template. This approach is inspired by natural biomineralization processes, and employs physiological processing conditions to enable inclusion of biologically active growth factors. Specific Aim 2 will engineer growth factors tagged with a putative mineral binding sequence for inclusion into mineral materials, and will systematically characterize growth factor inclusion, release, and biological activity. The emphasis will be on achieving a high level of control over release rates while maintaining biological activity. We will then use this approach to sequentially release two growth factors that influence MSC activity: a mitogenic factor (FGF-2) and an osteogenic factor (BMP-2). Specific Aim 3 will evaluate temporally controlled MSC activity within the mineral matrices developed in S.A.2, focusing on optimizing the presence of functional, differentiated osteoblasts. This aim is designed to demonstrate the utility of our approach in an application with substantial clinical significance: engineering of functional bone tissue. The results of this aim will serve as a springboard for development of an extensive research program focused on temporally controlling growth factor presentation to stem cells.